Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election with traverse of Group I (Claims 20-24 and 26; drawn to a method of producing oligodendrocytes) in the reply filed on July 12, 2026, is acknowledged.
Claims 25 and 27-28 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention (Group II), there being no allowable generic or linking claim.
Response to Arguments
Applicant's election with traverse of Group I is acknowledged. The traversal is on the ground(s) that the specific steps of the oligodendrocyte differentiation protocol as currently claimed in Groups I-II make a contribution over the cited prior art, US2017095512 (Izrael), and is, therefore, a special technical feature. Furthermore, 37 CFR 1.475(b)(3) identifies that a product, a process specially adapted for the manufacture of said product, and a use of the said product, are considered linked inventions that should be examined together without restriction. The method of Claim 20 and the treatment method of Claim 25 constitute a product-process-use combination that will be considered to have unity of invention, thus restriction between Groups I and II should not be required. As such, Groups I-II should be rejoined (page 6, paragraph 5-page 10, paragraph 2)
Applicant’s traversal has been considered and is found persuasive because Izrael does not teach the specific method steps as identified in the claims associated with Groups I-II.
However, WO2020243618 (Fossati) teaches a method of differentiating oligodendrocytes from iPSCs comprising culturing iPSCs in neural induction media containing the small molecules SB431542 10 μM (Stemgent) and LDN193189 250 nM and 100 nM all-trans-RA (the same neural induction media used by the Applicant (Example 1 and Table S1)) where DMEM/F12 with the addition of about 25 μg/ml insulin could be used as the base media from days 0-7. Media changes were performed daily.
On day 8, the cultured cells express Pax6 (a neural cell marker). Although Fossati doesn’t identify that they generated PAX6+/NESTIN+/SOX2+ NSCs, they do identify that their cells express PAX6 and Fossati uses the same neural induction media, the same culture time frame (7 days), and both start with human pluripotent stem cells. Therefore, it is reasonable to conclude that the PAX6+ neural stem cells of Fossati would also express NESTIN and SOX2, as identified by the instant application. On day 8, the media was switched to N2 medium comprising glutamax, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, N2 supplement, 100 nM retinoic acid, and 1 μM SAG. The cells were cultured with this medium composition through day 11. Fossati identified that SAG and purmorphamine are known alternative Smoothened agonists and would be considered interchangeable. Therefore, it would have been well understood that purmorphamine could be used in place of SAG.
On day 12, overconfluent cells were piling up and 3D structures were clearly visible and this is an important checkpoint before proceeding with the differentiation. Cells expressed OLIG2 and NKX2.2. At day 12, adherent cells were detached mechanically or enzymatically to allow for sphere formation. Cells were re-plated into Ultra-low attachment plates in N2B27 Medium (glutamax, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, N2 supplement, B27 supplement) containing 100 nM RA and 1 μM SAG, changing it every other day until day 20. As stated supra, Fossati identified that SAG and purmorphamine are known alternative Smoothened agonists and would be considered interchangeable. Therefore, it would have been well understood that purmorphamine could be used in place of SAG.
At day 20, medium was switched to PDGF Medium (glutamax, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, N2 supplement, B27 supplement, PDGF, IGF-1, HGF, NT3, Insulin, Biotin, cAMP, and T3), and 2/3 media changes were performed every other day until day 30. From day 12 to day 30, cells were grown as spheres.
At day 30, spheres were plated onto plates coated with poly-L-ornithine hydrobromide (50μg/mL) and Laminin (20μg/ml) at a density of 2 spheres/cm2 (about 20 spheres per well in a 6-vvell plate). This density was optimized to allow cells to migrate out from the sphere, proliferate and spread to the entire dish by the end of the protocol without the need for passaging. A p200 pipette was used to pick aggregates that were round, golden/brown with a dark center, having a diameter between 300 and 800μm. Spheres that were completely transparent were avoided, as these do not differentiate to oligodendrocytes. At this stage, plated spheres were cultured in a glial differentiation medium without any mitogen and lacking growth factors (Option B). Option B was developed to provide a shorter and less costly version of the protocol. O4+ staining occurred at day day 55 in option B. Additionally, 34 ± 4% of O4+ OPCs differentiated into MBP+ mature oligodendrocytes after growth-factor withdrawal from the medium for at least two weeks. MBP+ cells were observed beginning at about day 60 (day 30 of the culture step) (Figure 1, paragraphs 0068-0074, 0098-0129 and Table 2).
Fossati does not teach adding a Cannabinoid receptor agonist from day 8 to day 20 (steps 2-3).
However, Xapelli et al. ((Front.Cell.Neurosci. 8: 1-9. 2014)) teaches that it was previously found that Cannabinoid Receptor 1 and 2 (CB1R and CB2R are expressed in vivo in the SVZ and DG and in nestin+ cells from neural progenitor cultures. Moreover, endocannabinoids have emerged as a potential target to modulate oligodendrogenesis, by modulating CB1R and CB2R. Postnatal treatment with a CB1R agonist increased the number of Olig2+ cells in the rat SVZ. Neural stem cells (NSCs) from the SVZ can be highly expanded in culture and give rise to oligodendrocytes. Xapelli teaches that they incubated SVZ neurospheres with Hp (a CBR1 agonist; 100 nM or 1 μM) and found that Hp treatment did not affect cell death and proliferation, but promoted oligodendrocytic differentiation and maturation of SVZ stem/progenitor cells (page 2, column 1, paragraph 4-page 7, column 1, paragraph 3).
Similarly, Gomez et al. (J Neuroimmune Pharmacol 10: 309–317. 2015) teaches that a basal tone of the endocannabinoid 2 arachidonoylglycerol (2-AG; a CB1 and 2 Receptor agonist) enhances late oligodendrocyte progenitor cell (OPC) differentiation. Furthermore, they found that culturing OPCs with agonists of cannabinoid receptor CB1 (ACEA; 0.5 μM), CB2 (JWH133; 0.5 μM) or both (HU-210; 0.5 μM) significantly increased the number of Ki67+ Olig2+ proliferating OPCs (whole document).
Tomas-Roig et al. (CNS Neuroscience & Therapeutics 22: 387–395. 2016) teaches that cannabinoid agonist WIN55212.2 (a CB1R and CB2R agonist) promotes oligodendrocyte differentiation in vitro when cultured with immature oligodendrocytes at a concentration of 1 μM (whole document).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the iPSC to oligodendrocyte differentiation protocol of Fossati by adding the CB receptor agonist WIN55212.2 to the media from days 8-20 to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because Xapelli teaches that Postnatal treatment with a CB1R agonist increased the number of Olig2+ cells in the rat SVZ (where neural stem cells are found) and that culturing neural stem cells (the starting cell population of stage 2) with Hp (a CBR1 agonist; 100 nM or 1 μM) promoted oligodendrocytic differentiation and maturation of SVZ stem/progenitor cell while not negatively affecting cell death and proliferation. Furthermore, Gomez teaches that culturing OPCs with agonists of cannabinoid receptor CB1R (ACEA; 0.5 μM), CB2R (JWH133; 0.5 μM) or both (HU-210; 0.5 μM) significantly increased the number of Ki67+ Olig2+ proliferating OPCs. Therefore, it would have been obvious to one of ordinary skill that they could add agonists of the cannabinoid receptors to the media during neural stem cell differentiation (days 8-11) and oligodendrocyte progenitor cell (OPC) differentiation culture stages (days 12-20) to improve the differentiation of NSCs to OPCs and to increase the proliferative capacity of Olig2+ OPCs during days 12-20, increasing the number of OPCs that can be further differentiated into oligodendrocytes at later stages of the differentiation protocol which should increase the overall yield of oligodendrocytes. Regarding using WIN55212.2 as the agonist, Tomas-Roig identifies that WIN55212.2 is a known CB1R and CB2R agonist that has been shown to improve oligodendrocyte differentiation and, as stated supra, Xapelli and Gomez identify that CB1R and CB1R and CB2R agonists promote oligodendrocytic differentiation and maturation of SVZ stem/progenitor cell and the proliferative capacity of Olig2+ OPCs. Therefore, it would have well understood that WIN55212.2, a known CB1R and CB2R agonist, could be added to the NSC and OPC culture stages as the CB receptor agonist as it was a known alternative. Regarding the concentration of the WIN55212.2 used for the culturing steps, Xapelli uses concentrations of 100nM and 1 μM, Gomez used 0.5 μM, and Tomas-Roig used 1 μM. As each of the art used similar concentrations and Xapelli and Tomas-Roig identify that 1 μM is safe, it would have been well understood that 1 μM of WIN55212.2 could be used for both culture stages. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
As such, the method of differentiating iPSCs into oligodendrocytes of Groups I-II is not considered a special technical feature, as argued. Therefore, the restriction is maintained.
DETAILED ACTION
The amended claims filed on July 12, 2026, have been acknowledged. Claims 1-19 were cancelled. Claims 20-25 were amended. Claims 27-28 are new. In light of the Applicant’s elected species, claims 25 and 27-28 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Claims 20-24 and 26 are pending and examined on the merits.
Priority
Acknowledgment is made of Applicant’s claim for foreign priority under 35 U.S.C. 119(a)-(d).The applicant claims foreign priority from CN202210194839.8 filed on March 2, 2022. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55, received November 14, 2023. While a certified copy of the foreign patent application CN202210194839.8 is provided with the instant application, a certified English translation of said foreign patent application has not been provided.
Information Disclosure Statement
The information disclosure statement (IDS) filed on November 14, 2023, has been considered.
Claim Objections
Claim 20 is objected to because of the following informalities:
instant claim uses the abbreviation “NSCs”, which has not been spelled out upon first use. Similarly, instant claim uses the abbreviation “CB” and “PDGF-AA”, which has not been spelled out upon first use. Although claims are allowed abbreviations, if an abbreviation is not spellout upon first use in a claim, MPEP §2429 states that Applicant only use abbreviations that are specifically defined in "WIPO Standard ST.25 (1998)" or that are well known and would be clear to someone who had not read the invention description.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 20-24 and 26 are rejected under 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph, as based on a disclosure which is not enabling. The disclosure does not enable one of ordinary skill in the art to practice the invention without identifying cells used in the individual culture steps in the step-wise differentiation of pluripotent stem cells into oligodendrocytes, which is/are critical or essential to the practice of the invention but not included in the claim(s). See In re Mayhew, 527 F.2d 1229, 188 USPQ 356 (CCPA 1976).
The factors to be weighed to evaluate whether a disclosure satisfies the enablement requirement and whether any necessary experimentation is undue are set forth in MPEP 2164.01(a).
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
Although all the factors have been considered, the relevant factors will be addressed below.
Breadth of the claims: Claim 20 recites the following claim language, “A method for producing oligodendrocytes, comprising: (1) generating NSCs of PAX6+/NESTIN+/SOX2+: human induced pluripotent stem cells are cultured with neural induction medium from day 0 to day 7, and the medium is changed every day; (2) from day 8, changing the medium to fresh N2 medium containing retinoic acid, Purmorphamine and CB receptor agonist every day; (3) from day 12, manually isolating the cells and suspending them in N2B27 medium containing retinoic acid, Purmorphamine and CB receptor agonist, and changing the medium every other day; (4) from day 20, further suspending spheroids in PDGF-AA medium; (5) from day 30, seeding and culturing expanded spheroids on Matrigel-coated plates or Petri dishes with low growth factors in glial differentiation medium for 16-36 days; wherein the concentration of the retinoic acid is 10-1000nM, the concentration of the Purmorphamine is 0.1-10 μM, and the concentration of the CB receptor agonist is 0.1-10 μM in the medium of said (2) and (3), wherein the CB receptor agonist is WIN55212-2”. The broadest reasonable interpretation is that this method could be done with any starting induced pluripotent stem cell (iPSC) or any PAX6+/NESTIN+/SOX2+ NSCs produced from any method (claim 21) and using any spheroids for stages 4 and 5 as there is no previously recited step of generating spheroids or recitation of a spheroid related to steps 1-3.
Nature of the invention: The subject matter of the invention relates to a method to differentiate pluripotent stem cells into oligodendrocytes using the stepwise differentiation protocol as recited in claims 20-21.
State of the prior art: The prior art teaches that a multi-step procedure is required for generating oligodendrocytes from induced pluripotent stem cells and that the type of cell being cultured is an important consideration for achieving differentiation of stem cells to oligodendrocytes, as identified by World Intellectual Property Organization Patent Application No. 2020243618 (Fossati).
Fossati teaches a method of differentiating oligodendrocytes from iPSCs comprising culturing iPSCs in neural induction media containing the small molecules SB431542 10 μM (Stemgent) and LDN193189 250 nM and 100 nM all-trans-RA (the same neural induction media used by the Applicant (Example 1 and Table S1)) where DMEM/F12 with the addition of about 25 μg/ml insulin could be used as the base media from days 0-7. Media changes were performed daily.
On day 8, the cultured cells express Pax6 (a neural cell marker). Although Fossati doesn’t identify that they generated PAX6+/NESTIN+/SOX2+ NSCs, they do identify that their cells express PAX6 and Fossati uses the same neural induction media, the same culture time frame (7 days), and both start with human pluripotent stem cells. Therefore, it is reasonable to conclude that the PAX6+ neural stem cells of Fossati would also express NESTIN and SOX2, as identified by the instant application. On day 8, the media was switched to N2 medium comprising glutamax, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, N2 supplement, 100 nM retinoic acid, and 1 μM SAG. The cells were cultured with this medium composition through day 11. Fossati identified that SAG and purmorphamine are known alternative Smoothened agonists.
On day 12, overconfluent cells were piling up and 3D structures were clearly visible and this is an important checkpoint before proceeding with the differentiation. Cells expressed OLIG2 and NKX2.2. At day 12, adherent cells were detached mechanically or enzymatically to allow for sphere formation. Cells were re-plated into Ultra-low attachment plates in N2B27 Medium (glutamax, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, N2 supplement, B27 supplement) containing 100 nM RA and 1 μM SAG, changing it every other day until day 20. As stated supra, Fossati identified that SAG and purmorphamine are known alternative Smoothened agonists.
At day 20, medium was switched to PDGF Medium (glutamax, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, N2 supplement, B27 supplement, PDGF, IGF-1, HGF, NT3, Insulin, Biotin, cAMP, and T3), and 2/3 media changes were performed every other day until day 30. From day 12 to day 30, cells were grown as spheres.
At day 30, spheres were plated onto plates coated with poly-L-ornithine hydrobromide (50μg/mL) and Laminin (20μg/ml) at a density of 2 spheres/cm2 (about 20 spheres per well in a 6-vvell plate). This density was optimized to allow cells to migrate out from the sphere, proliferate and spread to the entire dish by the end of the protocol without the need for passaging. A p200 pipette was used to pick aggregates that were round, golden/brown with a dark center, having a diameter between 300 and 800μm. Spheres that were completely transparent were avoided, as these do not differentiate to oligodendrocytes. At this stage, plated spheres were cultured in a medium containing mitogen (Option A) or in a glial differentiation medium without any mitogen and lacking growth factors (Option B). Option A was optimized to obtain the highest yield of O4+ cells, while Option B was developed to provide a shorter and less costly version of the protocol. O4+ staining occurred at day 75 in option A and day 55 in option B. Additionally, 34 ± 4% of O4+ OPCs differentiated into MBP+ mature oligodendrocytes after growth-factor withdrawal from the medium for at least two weeks. MBP+ cells were observed beginning at about day 60 (day 30 of the culture step) (Figure 1, paragraphs 0068-0074, 0098-0129 and Table 2).
The prior art identifies that cannabinoid receptor agonists are known to be involved in multiple stages of the differentiation of neural stem cells into oligodendrocyte progenitor cells and oligodendrocytes, as identified by Xapelli et al. (Front.Cell.Neurosci. 8: 1-9. 2014), Gomez et al. (J Neuroimmune Pharmacol 10: 309–317. 2015), and Tomas-Roig et al. (CNS Neuroscience & Therapeutics 22: 387–395. 2016).
Xapelli teaches that it was previously found that Cannabinoid Receptor 1 and 2 (CB1R and CB2R are expressed in vivo in the SVZ and DG and in nestin+ cells from neural progenitor cultures. Moreover, endocannabinoids have emerged as a potential target to modulate oligodendrogenesis, by modulating CB1R and CB2R. Postnatal treatment with a CB1R agonist increased the number of Olig2+ cells in the rat SVZ. Neural stem cells (NSCs) from the SVZ can be highly expanded in culture and give rise to oligodendrocytes. Xapelli teaches that they incubated SVZ neurospheres with Hp (a CBR1 agonist; 100 nM or 1 μM) and found that Hp treatment did not affect cell death and proliferation, but promoted oligodendrocytic differentiation and maturation of SVZ stem/progenitor cells (page 2, column 1, paragraph 4-page 7, column 1, paragraph 3).
Similarly, Gomez teaches that a basal tone of the endocannabinoid 2 arachidonoylglycerol (2-AG; a CB1 and 2 Receptor agonist) enhances late oligodendrocyte progenitor cell (OPC) differentiation. Furthermore, they found that culturing OPCs with agonists of cannabinoid receptor CB1 (ACEA; 0.5 μM), CB2 (JWH133; 0.5 μM) or both (HU-210; 0.5 μM) significantly increased the number of Ki67+ Olig2+ proliferating OPCs (whole document).
Tomas-Roig teaches that cannabinoid agonist WIN55212.2 (a CB1R and CB2R agonist) promotes oligodendrocyte differentiation in vitro when cultured with immature oligodendrocytes at a concentration of 1 μM (whole document).
As such, the prior art shows that a specific cell type are used for each stage of differentiation: iPSCs in stage 1, Pax6+ neural stem cells in stage 2, OLIG2+ and NKX2.2+ cells with 3D structures at stages 3-4, round, golden/brown OPC cell aggregates with a dark center at stage 5, O4+ mature OPCs cells for stage 6 differentiation of OPCs into oligodendrocytes. Furthermore, Fossati clearly identifies multiple important considerations for proper differentiation of the iPSCs into oligodendrocytes as 3D structures are important to see before proceeding with stages 3-4; OPC spheres with golden/brown with a dark center are important as spheres that were completely transparent were avoided, as these do not differentiate to oligodendrocytes; and O4+ cells are important for differentiating OPCs into oligodendrocytes. Therefore, careful consideration of the cells associated with each differentiation step is important for successful differentiation of stem cells into oligodendrocytes.
Level of predictability in the art: The prior art has successfully reduced to practice that a multistep differentiation protocol with refined control of concentration, time and duration of treatment with the defined growth and differentiation factors is required to generate oligodendrocyte cells from pluripotent stem cells. As such, this results in unpredictability about how someone can use this method to differentiate induced pluripotent stem cells into oligodendrocytes without defining the cell types used with each media composition of each step of the multistep differentiation protocol of claims 20 and 21.
Amount of direction provided by the inventor and existence of working examples: The Applicant discloses that they used a defined protocol for differentiating induced pluripotent stem cell types into oligodendrocytes.
The human induced pluripotent stem cell (hiPSC) line (UE017C1) was cultured on Matrigel-coated six-well plates using mTeSRTM. In order to generate PAX6+/NESTIN+/SOX2+ NSC, the dual SMAD neural induction method was used. hiPSCs with a density of -80% with neural induction medium (NIM) were treated from day 0 (DO) to day 7, and the medium was changed every day. Starting from D8, the medium was changed daily to fresh N2 medium containing RA (100nM), purmorphamine (PUR, 1 μM) and WIN (1 μM). Starting from D12, cells were manually isolated and suspended in the ultra-low attachment plate in N2B27 medium containing RA (100 nM), PUR (1 μM) and WIN (1 μM), and the medium was changed every other day. Starting from D20, the spheroids were further suspended in PDGF-AA medium for culture. At D30, expanded spheroids were seeded on Matrigel-coated plates or Petri dishes with low growth factors in the glial differentiation medium (GDM). All medium and their compositions are listed in Table S1. Fig. 1 shows the timeline of the culture process of the present invention.
It should be understood that the PAX6+/NESTIN+/SOX2+ NSC described in the present invention is not limited to those obtained by induced differentiation with hiPSCs, but can also be directly obtained from commercially available products, and further cultured according to the method described in the present invention to obtain oligodendrocyte precursor cells (OPCs) and oligodendrocytes (OLs) for the treatment of spinal cord injury (SCI) (Example 1).
Addition of WIN (1 μM) during stages 2 and 3 increased the production of OLIG2+ cells, OLIG2+ spheroids, PDGFRα expression in OPCs at day 34 of culture, and O4+/MBP+ oligodendrocytes compared to control culture conditions. Furthermore, Applicant identifies that OLIG2- cells failed to form spheroids (Figures 3-4 and 6 and page 6, line 8-page 8, line 23).
As can be seen in Applicant’s examples, the differentiation protocol follows a specific differentiation procedure beginning with iPSCs being cultured with the neural induction medium and proceeding from there to generate oligodendrocytes expressing 04 and MBP.
Quantity of experimentation needed: In light of the above factors, the prior art and the Applicant disclose closely related multi-step differentiation protocols and that the cell type being added and the media composition are important considerations for each step of the differentiation protocol. Therefore, a defined differentiation protocol is necessary to generate oligodendrocyte cells from pluripotent stem cells. For example, Fossati and the Applicant identify that OLIG2 expression is important before proceeding with further differentiation as the Applicant identifies that OLIG2- cells fail to form spheroids. Furthermore, Fossati identifies that at stage 5 (day 30 and on), spheres that were completely transparent were avoided, as these do not differentiate to oligodendrocytes. As such, essential steps are considered to be missing from claim 20. There would be undue experimentation related to any culturing method that does not have a defined multistep differentiation protocol in line with the prior art and the Applicant’s specification to practice the full scope of the claim.
Claims 21-24 and 26 are also rejected because of their dependency on claim 20.
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 20-24 and 26 rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 20 recites the limitations "suspending spheroids" and “expanding spheroids” in lines 10 and 11, respectively. There is insufficient antecedent basis for this limitation in the claim. There is no previous recitation of spheroids. As such, it is unclear what spheroids are being referenced. It is presumed that stage 3 will involve spheroid formation. However, as this has not been explicitly stated, it is unclear if this is what is meant by the spheroids.
Claims 21-24 and 26 are also rejected because of their dependency on claim 20.
The term “low growth factors” in claim 20 is a relative term which renders the claim indefinite. The term “low growth factors” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is not clear what amount of growth factors would fall under low levels.
Claims 21-24 and 26 are also rejected because of their dependency on claim 20.
Claim 21 recites the limitation "the finished NSC product" in line 2. There is insufficient antecedent basis for this limitation in the claim. There is no previous recitation of a finished NSC product nor any method of making an NSC product outside of the one identified in stage 1 of claim 20, which claim 21 specifically replaces. As such, it is unclear what finished product and what method of generating the finished product is being referenced.
Claim 23 is also rejected because of its dependence on claim 21.
Claims 22-23 contain the trademarks/trade names “Matrigel” and “mTESR”. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademarks/trade names are used to identify/describe a mixture of extracellular matrix proteins (Matrigel) and a pluripotent stem cell culture media (mTESR) and, accordingly, the identification/description is indefinite.
Claim 23 recites the limitation "the human induced pluripotent stem cells" in line 2. There is insufficient antecedent basis for this limitation in the claim. There is no previous recitation of a human induced pluripotent stem cell in relation to the finished NSC product as no method of making the NSC product is recited outside of the one identified in stage 1 of claim 20, which claim 21 specifically replaces. As such, it is unclear what method of generating the finished product is used and whether human iPSCs are used to generate the finished product.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 21 and 23 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 21 recites the limitation "wherein said (1) is replaced with the finished NSC product". Claim 20 requires the step of (1). Therefore, it cannot be replaced in claim 21 without failing to include all the limitations of claim 20 upon which it depends.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 20-24 and 26 are rejected under 35 U.S.C. 103 as being unpatentable over World Intellectual Property Organization Patent Application No. 2020243618 (Fossati) and further in view of Xapelli et al. (Front.Cell.Neurosci. 8: 1-9. 2014), Gomez et al. (J Neuroimmune Pharmacol 10: 309–317. 2015; referenced in IDS), and Tomas-Roig et al. (CNS Neuroscience & Therapeutics 22: 387–395. 2016; referenced in IDS).
Regarding claims 20, 24, and 26, Fossati teaches a method of differentiating oligodendrocytes from iPSCs comprising culturing iPSCs in neural induction media containing the small molecules SB431542 10 μM (Stemgent) and LDN193189 250 nM and 100 nM all-trans-RA (the same neural induction media used by the Applicant (Example 1 and Table S1)) where DMEM/F12 with the addition of about 25 μg/ml insulin could be used as the base media from days 0-7. Media changes were performed daily.
On day 8, the cultured cells express Pax6 (a neural cell marker). Although Fossati doesn’t identify that they generated PAX6+/NESTIN+/SOX2+ NSCs, they do identify that their cells express PAX6 and Fossati uses the same neural induction media, the same culture time frame (7 days), and both start with human pluripotent stem cells. Therefore, it is reasonable to conclude that the PAX6+ neural stem cells of Fossati would also express NESTIN and SOX2, as identified by the instant application. On day 8, the media was switched to N2 medium comprising glutamax, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, N2 supplement, 100 nM retinoic acid, and 1 μM SAG. The cells were cultured with this medium composition through day 11. Fossati identified that SAG and purmorphamine are known alternative Smoothened agonists and would be considered interchangeable. Therefore, it would have been well understood that purmorphamine could be used in place of SAG.
On day 12, overconfluent cells were piling up and 3D structures were clearly visible and this is an important checkpoint before proceeding with the differentiation. Cells expressed OLIG2 and NKX2.2. At day 12, adherent cells were detached mechanically or enzymatically to allow for sphere formation. Cells were re-plated into Ultra-low attachment plates in N2B27 Medium (glutamax, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, N2 supplement, B27 supplement) containing 100 nM RA and 1 μM SAG, changing it every other day until day 20. As stated supra, Fossati identified that SAG and purmorphamine are known alternative Smoothened agonists and would be considered interchangeable. Therefore, it would have been well understood that purmorphamine could be used in place of SAG.
At day 20, medium was switched to PDGF Medium (glutamax, non-essential amino acids, β-mercaptoethanol, penicillin-streptomycin, N2 supplement, B27 supplement, PDGF, IGF-1, HGF, NT3, Insulin, Biotin, cAMP, and T3), and 2/3 media changes were performed every other day until day 30. From day 12 to day 30, cells were grown as spheres.
At day 30, spheres were plated onto plates coated with poly-L-ornithine hydrobromide (50μg/mL) and Laminin (20μg/ml) at a density of 2 spheres/cm2 (about 20 spheres per well in a 6-vvell plate). This density was optimized to allow cells to migrate out from the sphere, proliferate and spread to the entire dish by the end of the protocol without the need for passaging. A p200 pipette was used to pick aggregates that were round, golden/brown with a dark center, having a diameter between 300 and 800μm. Spheres that were completely transparent were avoided, as these do not differentiate to oligodendrocytes. At this stage, plated spheres were cultured in a glial differentiation medium without any mitogen and lacking growth factors (Option B). Option B was developed to provide a shorter and less costly version of the protocol. O4+ staining occurred at day day 55 in option B. Additionally, 34 ± 4% of O4+ OPCs differentiated into MBP+ mature oligodendrocytes after growth-factor withdrawal from the medium for at least two weeks. MBP+ cells were observed beginning at about day 60 (day 30 of the culture step) (Figure 1, paragraphs 0068-0074, 0098-0129 and Table 2).
Fossati does not teach adding a Cannabinoid receptor agonist from day 8 to day 20 (steps 2-3).
However, Xapelli teaches that it was previously found that Cannabinoid Receptor 1 and 2 (CB1R and CB2R are expressed in vivo in the SVZ and DG and in nestin+ cells from neural progenitor cultures. Moreover, endocannabinoids have emerged as a potential target to modulate oligodendrogenesis, by modulating CB1R and CB2R. Postnatal treatment with a CB1R agonist increased the number of Olig2+ cells in the rat SVZ. Neural stem cells (NSCs) from the SVZ can be highly expanded in culture and give rise to oligodendrocytes. Xapelli teaches that they incubated SVZ neurospheres with Hp (a CBR1 agonist; 100 nM or 1 μM) and found that Hp treatment did not affect cell death and proliferation, but promoted oligodendrocytic differentiation and maturation of SVZ stem/progenitor cells (page 2, column 1, paragraph 4-page 7, column 1, paragraph 3).
Similarly, Gomez teaches that a basal tone of the endocannabinoid 2 arachidonoylglycerol (2-AG; a CB1 and 2 Receptor agonist) enhances late oligodendrocyte progenitor cell (OPC) differentiation. Furthermore, they found that culturing OPCs with agonists of cannabinoid receptor CB1 (ACEA; 0.5 μM), CB2 (JWH133; 0.5 μM) or both (HU-210; 0.5 μM) significantly increased the number of Ki67+ Olig2+ proliferating OPCs (whole document).
Tomas-Roig teaches that cannabinoid agonist WIN55212.2 (a CB1R and CB2R agonist) promotes oligodendrocyte differentiation in vitro when cultured with immature oligodendrocytes at a concentration of 1 μM (whole document).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the iPSC to oligodendrocyte differentiation protocol of Fossati by adding the CB receptor agonist WIN55212.2 to the media from days 8-20 to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because Xapelli teaches that Postnatal treatment with a CB1R agonist increased the number of Olig2+ cells in the rat SVZ (where neural stem cells are found) and that culturing neural stem cells (the starting cell population of stage 2) with Hp (a CBR1 agonist; 100 nM or 1 μM) promoted oligodendrocytic differentiation and maturation of SVZ stem/progenitor cell while not negatively affecting cell death and proliferation. Furthermore, Gomez teaches that culturing OPCs with agonists of cannabinoid receptor CB1R (ACEA; 0.5 μM), CB2R (JWH133; 0.5 μM) or both (HU-210; 0.5 μM) significantly increased the number of Ki67+ Olig2+ proliferating OPCs. Therefore, it would have been obvious to one of ordinary skill that they could add agonists of the cannabinoid receptors to the media during neural stem cell differentiation (days 8-11) and oligodendrocyte progenitor cell (OPC) differentiation culture stages (days 12-20) to improve the differentiation of NSCs to OPCs and to increase the proliferative capacity of Olig2+ OPCs during days 12-20, increasing the number of OPCs that can be further differentiated into oligodendrocytes at later stages of the differentiation protocol which should increase the overall yield of oligodendrocytes. Regarding using WIN55212.2 as the agonist, Tomas-Roig identifies that WIN55212.2 is a known CB1R and CB2R agonist that has been shown to improve oligodendrocyte differentiation and, as stated supra, Xapelli and Gomez identify that CB1R and CB1R and CB2R agonists promote oligodendrocytic differentiation and maturation of SVZ stem/progenitor cell and the proliferative capacity of Olig2+ OPCs. Therefore, it would have well understood that WIN55212.2, a known CB1R and CB2R agonist, could be added to the NSC and OPC culture stages as the CB receptor agonist as it was a known alternative. Regarding the concentration of the WIN55212.2 used for the culturing steps, Xapelli uses concentrations of 100nM and 1 μM, Gomez used 0.5 μM, and Tomas-Roig used 1 μM. As each of the art used similar concentrations and Xapelli and Tomas-Roig identify that 1 μM is safe, it would have been well understood that 1 μM of WIN55212.2 could be used for both culture stages. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding claim 21, due to the lack of clarity associated with what defines the finished product and the fact that dependent claim 23 identifies differentiating human iPSCs as a potential source of the NSC finished product, the method of Fossati differentiating human iPSCs into NSCs that express PAX6 is considered to read on claim 21.
Regarding claims 22-23, Fossati teaches that as part of their differentiation protocol, the pluripotent stem cells were plated on Matrigel at a density of 10x 103 cells/cm2 in mTeSR1 medium. At all stages of differentiation cells are cultured in 5% CO2 incubators at 37°C (paragraphs 116-118 and 0183).
Conclusion
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/KEENAN A BATES/Examiner, Art Unit 1631
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